INTRODUCTION

Psittacine beak and feather disease (PBFD) is aviral disease that was first discovered in 1975 incockatoos in Australia. It is caused by the psittacinebeak and feather disease virus (PBFDV) which is asmall (14–18 nm diameter), icosahedral, non-enveloped virus with a circular single-strandedDNA (ssDNA) genome that has been characterisedand placed in the family Circoviridae (Raidal, Firth& Cross 1993; Studdert 1993; Pass, Plant & Sexton1994; Sexton, Penhale, Plant & Pass 1994; Ramis,Latimer, Gibert & Campagnoli 1998; Ypelaar, Bas-sami, Wilcox & Raidal 1999; Todd 2000).

The disease occurs predominantly in sulphur-crest-ed cockatoos (Cacatua galerita) but also in budger-igars (Melopsittacus undulatus) and other psittacinebirds. Characteristic clinical signs include symmetricfeather dystrophy and loss and deformity of thebeak in some species (Ramis et al. 1998). Birdsinfected with PBFDV usually die as a result of sec-ondary infections, which support the view that theyare immunosuppressed; bursal and thymic lesionsare consistently detected (Todd 2000).

Histopathology has been used for the routine diag-nosis of PBFD, but more recently, polymerase chainreaction (PCR) and haemagglutination (HA) testshave been described for the detection of PBFDVand haemagglutination inhibition (HI) for the detec-tion of antibodies to PBFDV (Riddoch, Raidal, &Cross 1996). Since all attempts to cultivate PBFDV

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Onderstepoort Journal of Veterinary Research, 71:29–34 (2004)

Psittacine beak and feather disease virusin budgerigars and ring-neck parakeetsin South Africa

J. ALBERTYN, K.M. TAJBHAI and R.R. BRAGG*

Department of Microbial, Biochemical and Food Biotechnology, University of the Free StateP.O. Box 339, Bloemfontein, 9300 South Africa

ABSTRACT

ALBERTYN, J., TAJBHAI, K.M. & BRAGG, R.R. 2003. Psittacine beak and feather disease virus inbudgerigars and ring-neck parakeets in South Africa. Onderstepoort Journal of Veterinary Research,71:29–34

Psittacine beak and feather disease (PBFD) is a common disease of the psittacine species and iscaused by the psittacine beak and feather disease virus (PBFDV). In this study the occurrence of thedisease in ring-neck parakeets and budgerigars in South Africa suffering from feathering problems,using polymerase chain reaction as a diagnostic test was investigated. The genetic variation betweenviral isolates was also studied. Results indicate that PBFDV can be attributed to being the cause offeathering problems in some of the ring-neck parakeets and budgerigars in South Africa. Geneticvariation of isolates occurs between species and individuals. A cheap and easy to use method ofblood sample collection on filter paper for diagnostic purposes was also evaluated. It proved to beless stressful to the birds and did not inhibit further processes.

Keywords: Budgerigars, genetic variation, psittacine beak and feather disease virus, ring-neck para-keets, South Africa

* Author to whom correspondence is to be directed. E-mail:BraggRR@sci.uovs.ac.za

Accepted for publication 9 September 2003—Editor

have so far been unsuccessful, the diagnosis of thedisease must be confirmed by the demonstration ofviral antigen or viral nucleic acid (Todd 2000).

Bassami, Ypelaar, Berryman, Wilcox & Raidal(2001) studied the genetic diversity of PBFDV andconcluded that there was no evidence that distinct-ly different genotypes occur although various differ-ences due to small deletions or insertions are pres-ent in the genomes of the isolates. They stated thatthe significance of these differences is unknown butthat there is little evidence to support a relationshipbetween genetic variation and regional distributionof isolates, or that there are differences in patho-genicity, antigenicity or any physiochemical charac-teristics of the virus.

There is no known cure for PBFD but a vaccineconsisting of inactivated PBFDV in a double-oilemulsion adjuvant system (DOE vaccine) has beeninvestigated (Raidal et al. 1993; Raidal & Cross1994), and its use can be a safe and effective aidfor controlling the disease if it is combined with othermanagement procedures, such as good biosecurity.

In the disease, avian polyoma, caused by the avianpolyomavirus (APV), clinical and pathological signssimilar to those of PBFD are manifested. It has beenknown to occur in concurrent outbreaks with PBFDalthough documentation of these occurrences islimited (Latimer, Niagro, Campagnoli, Ritchie, Pesti& Steffens 1993; Ramis et al. 1998). Differentiationbetween the two diseases is difficult as the macro-and microscopical pathology of the two diseases issimilar; diagnosis of avian polyoma disease is per-formed using techniques such as DNA in situ hybrid-ization or PCR (Ramis et al. 1998).

Bird breeders in South Africa have been reportingfeathering problems in budgerigars and have sus-pected that this may be due to APV infection. How-ever, Bragg (2002) reported that PBFDV is wide-spread in parrots of South Africa breeders and sug-gested that the feathering problems in the budgeri-gars could be due to PBFDV infection and not APVinfection.

This study was undertaken to determine if the occur-rence of PBFD in budgerigars and ring-neck para-keets in South Africa exists using PCR as an iden-tification tool. It was also undertaken to determine ifthere is any genetic variation between the isolates.A simple method for collecting blood samples on fil-ter paper was also evaluated for its use in down-stream processes for example the extraction ofviral DNA followed by PCR.

MATERIALS AND METHODS

A total of seventeen birds were obtained frombreeders in South Africa that showed featheringproblems indicative of PBFDV infection. Blood sam-ples were collected in heparin lithium tubes from sixring-neck parakeets in Bloemfontein, five budgeri-gars in Pretoria and five in Cradock, South Africa(Table 1). In addition, the effectiveness of bloodsample collection on filter paper was evaluated. Astrip of Whatman No.1 filter paper of approximately5 mm in width and 40 mm in length was sterilizedby U.V. radiation. A wing vein of one ring-neck para-keet (P-BLM-4) was pricked with a sterile 0.33 mmdiameter needle and four separate strips of filterpaper was used to absorb blood resulting in thecoverage of a blood-soaked area of approximately5 x 5 mm in size at one end of the filter paper. Thefilter papers were air-dried vertically at room tem-perature followed by viral DNA extraction from thesamples after 24, 48, 72 and 96 h. In addition, ablood spot sample from a ring-neck parakeet inGeorge, South Africa (P-GEO) that was submittedon a commercially available specimen collectionpaper was similarly processed after one month ofstorage at –20°C.

Viral DNA was extracted from the blood samplesusing the QIAamp® DNA Blood Mini Kit and driedblood spot samples were processed using theQIAamp® DNA Mini Kit according to the manufac-turer’s instructions (Qiagen). The PCR amplifica-

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Psittacine beak and feather disease virus in South Africa

TABLE 1 Blood samples obtained from species and sources

Sample Species Source

P-BLM-1 Parakeet BloemfonteinP-BLM-2 Parakeet BloemfonteinP-BLM-3 Parakeet BloemfonteinP-BLM-4 Parakeet BloemfonteinP-BLM-5 Parakeet BloemfonteinP-BLM-6 Parakeet BloemfonteinP-GEO Parakeet GeorgeB-PTA-1 Budgerigar PretoriaB-PTA-2 Budgerigar PretoriaB-PTA-3 Budgerigar PretoriaB-PTA-4 Budgerigar PretoriaB-PTA-5 Budgerigar PretoriaB-CRA-1 Budgerigar CradockB-CRA-2 Budgerigar CradockB-CRA-3 Budgerigar CradockB-CRA-4 Budgerigar CradockB-CRA-5 Budgerigar Cradock

tion was performed using a set of oligonucleotideprimers [PB F1 (AACCCTACAGACGGCGAG-3’)and PB R1 (5’-GTCACAGTCCTCCTTGTACC-3’)]obtained from literature (Ypelaar et al. 1999;Bassami et al. 2001), amplifying a region of openreading frame 1 with a predicted size of 717 bp.Amplification was carried out according to Ypelaaret al. (1999) with a slight modification as follows;denaturation was carried out at 94 °C and anneal-ing was performed at 55 °C. A second set of prim-ers [PB F2 (5’-AACCATGCCGTCCAAGGA-3’) andPB R2 (5’-TATCAGTAATTGATGGGGTGGG-3’)]was designed to amplify the complete region ofOpen Reading Frame 1 of the PBFDV genome(GENBANK, Accession number AF080560) result-ing in an amplicon with a predicted size of 876 bp.

Amplified products were identified by electrophore-sis on 0.8 % agarose gels and visualized under UVillumination. Both primer sets produced ampliconsof the expected size and those obtained with theprimer set PB F1 and PB R1 were used in subse-quent reactions. Restriction digests were performedto determine if there was any genetic differencebetween the isolates by means of digestion with therestriction endonuclease HaeIII. The digest prod-ucts were identified by electrophoresis on 2.5% lowmelting point agarose gels and visualized under UVillumination together with a 50 bp step ladder (Pro-mega) as a molecular weight marker. Due to thepossibility of different amplified products from sam-ples B-CRA-1, B-CRA-2, B-CRA-4 and B-CRA-5,these amplicons were cloned into pGEM™ TEasyVector System (Promega), small scale plasmid iso-lation were performed, the inserts removed from

two randomly selected clones and digested withHaeIII. The products were again visualized by elec-trophoresis.

RESULTS

All samples obtained from the ring-neck parakeetsand budgerigars (except for sample B-CRA-3) pro-duced PCR amplicons of the expected size ofapproximately 700 bp, using primer set PB F1 andPB R1. Sample B-CRA-3, that consistently pro-duced a negative result, was further used as a neg-ative control The positive samples amplified withprimer set PB F2 and PB R2 also produced frag-ments of the expected size of approximately 850 bp(data not shown).

Amplicons obtained using primer set PB F1 and PBR1 were submitted to restriction digestion usingHaeIII. The digest profile was similar for all the ring-neck parakeet samples obtained from Bloemfonteinwith approximate sizes of 450 bp, 250 bp and athird smaller fragment of approximately 50 bp (Fig.1, lanes 2–7). The profile for sample P-GEO dif-fered in size from the other ring-neck parakeetsamples yielding fragments of approximate sizes460 bp, 250 bp and less than 50 bp (Fig. 1, lane 8).

Samples obtained from the budgerigars in Pretoria(B-PTA-1, B-PTA-2, B-PTA-3, and B-PTA-5) hadsimilar digest profiles with fragments of approxi-mately 410 bp, 225 bp and 50 bp in size (Fig. 2,lanes 2–4, 6), except for an additional fragmentobserved in sample B-PTA-4 with an approximatesize of 370 bp (Fig. 2, lane 5).

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J. ALBERTYN, K.M. TAJBHAI & R.R. BRAGG

450 bp

250 bp

150 bp

50 bp

1 2 3 4 5 6 7 8 9

FIG. 1 Restriction digest profile forthe samples obtained fromring-neck parakeets usingthe restriction enzymeHaeIII. The digest productswere loaded onto a 2.5 %agarose gel together with a50 bp step ladder (Promega)[Lanes 1 and 9]. Samples P-BLM-1, P-BLM-2, P-BLM-3,P-BLM-4, P-BLM-5, P-BLM-6 and P-GEO were loadedin lanes 2–8 respectively.Arrows on the right handside indicate the discrepan-cy found of the ~50 bp bandbetween the six samplesobtained from Bloemfontein(upper arrow) in comparisonto the sample obtained fromGeorge (lower arrow)

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Psittacine beak and feather disease virus in South Africa

1 2 3 4 5 6 7

450 bp

250 bp

150 bp

50 bp

FIG. 2 Restriction digest profile for thesamples obtained from budgeri-gars (Pretoria) using the restric-tion enzyme HaeIII. The digestproducts were loaded onto a 2.5% agarose gel together with a50 bp step ladder (Promega)[Lanes 1 and 7]. The samples B-PTA-1, B-PTA-2, B-PTA-3, B-PTA-4 and B-PTA-5 (lanes 2–6respectively) gave similar pro-files with sample B-PTA-4 con-taining an additional fragment ofapproximately 370 bp in size.The fragments below the 50 bpmarker were light in intensityand therefore more difficult toobserve

1 2 3 4 5 6 7 8 9 10

450 bp

250 bp

150 bp

50 bp

1 2 3 4 5 6

450 bp

250 bp

150 bp

50 bp

FIG. 3 Restriction digest profile for thesamples obtained from budgeri-gars (Cradock) using the restric-tion enzyme HaeIII showing mul-tiple fragments. The digest prod-ucts were loaded onto a 2.5 %agarose gel together with a 50bp step ladder (Promega)[Lanes 1 and 6] as a molecularmass marker. The samples B-CRA-1, B-CRA-2, B-CRA-4 andB-CRA-5 were loaded in lanes2–5 respectively

FIG. 4 Restriction digest profile for tworandomly picked clones fromeach sample obtained frombudgerigars (Cradock) using therestriction enzyme HaeIII. Thedigest products were loadedonto a 2.5% agarose gel togeth-er with a 50 bp step ladder (Pro-mega) [Lanes 1 and 10] as amolecular mass marker. Load-ing was performed as follows, B-CRA-1 [lanes 2 and 3], B-CRA-2[lanes 4 and 5] B-CRA-4 [lanes6 and 7] and B-CRA-5 [lanes 8and 9]

The digest profile obtained for the samples obtainedfrom budgerigars in Cradock continuously resultedin an unusual profile (Fig. 3) that led to the possi-bility that more than one PCR amplicon (i.e. ampli-cons from genetically different viral isolates) ofapproximately 700 bp was present.

These amplicons were cloned into the vectorpGEM™ TEasy, inserts were purified from two ran-domly selected clones for each sample, and digest-ed with HaeIII (Fig. 4). Clones from B-CRA-1 (Fig.4, lanes 2 and 3) were similar in profile to eachother but different from B-CRA-2 and B-CRA-4 [Fig.4, lanes 4–7 respectively], which in turn shared asimilar profile. Sample B-CRA-5 [Fig. 4, lanes 8 and9] had a different profile for each clone; with oneclone (Fig. 4, lane 8) sharing a similar profile withsamples B-CRA-2 and B-CRA 4 and the secondclone (Fig. 4, lane 9) sharing a profile with that of B-CRA-1 This profile indicates that at least two genet-ically different PBFDV isolates were present inthese birds and, more importantly, that at least onebird contained both of these genetically differentisolates.

The samples collected on filter paper all producedamplicons of the expected size (approximately 700bp) and all amplicons, including the blood samplethat was processed after one month of storage at–20°C, showed the same intensity (Fig. 5). None ofthe products showed any non-specific amplificationindicating that the filter paper collection methods isan easy and cost effective way to collect and pro-cess blood samples.

DISCUSSION

The PCR primer set PB F1 and PB R1 and primerset PB F2 and PB R2 amplified the expected prod-

ucts in all the samples tested except for onebudgerigar that tested negative. The PCR ampli-cons obtained by amplification using primer set PBF2 and PB R2 were not used in further processesalthough this primer set also resulted in reproduc-ible results. The reason for designing these primersand performing amplifications using them was toattempt to amplify the entire ORF1, which will beimportant for future studies and the potential devel-opment of a subunit vaccine.

The variation in the digest profiles between speciesand individuals suggests genetic variation in thesequences of the amplified region but it is still un-known if these variances indicate any differences inantigenicity or pathogenicity. Profiles for all thesamples obtained from the different regions in thecountry were not similar to each other even thoughthey were from the same species (budgerigars fromPretoria and Cradock). There may be a possibilitythat there is a relationship between regional distri-bution and genetic variation but this study does notgive enough evidence to confirm or refute this.

Bassami et al. (2001) reported that there is a pos-sibility that adaptation of particular genotypes tospecific species may occur and that regional differ-ences in strains may develop; they suggested thatthese differences would have a significant implica-tion of the development of a universal PCR assaythat would detect all strains of PBFDV. The genet-ic variation between these birds lends an importantview in the production of a vaccine. If there are sig-nificant differences in the antigenicity or patho-genicity and other physiochemical characteristicsof these isolates then it is important to develop avaccine that can effectively protect a bird againstany isolate or isolates that may infect it at any par-

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J. ALBERTYN, K.M. TAJBHAI & R.R. BRAGG

1 2 3 4 5 6 7 8

450 bp

250 bp

150 bp

50 bp

FIG. 5 PCR amplified products of PBFDV (P-BLM-4) from samples collected on fil-ter paper, followed by DNA extractionafter 24 [lane 2], 48 [lane 3], 72 [lane4] and 96 h [lane 5]. Lane 6 containsthe PCR product of a sample (P-GEO) that was received on commer-cially available specimen collectionpaper and processed after 1 month.Lanes 1 and 8, contains a molecularmass marker (λ EcoR1/HindIII), andlane 7 a negative control from a sam-ple that was shown to be negative (B-CRA-3)

ticular time. In the related porcine circovirus (PCV)two distinct genotypes have been identified (Todd2000) and it is possible that similar genotypic dif-ferences have occurred in PBFDV.

The PCR tests performed were reproducible andconfirm the occurrence of PBFDV infection in thering-neck parakeets and budgerigars in the regionsfrom where the samples were obtained. However, itwas difficult to rule out the possibility of APV infec-tion in the birds tested. Although a PCR test usingprimers to amplify the region coding for protein VPIof APV was performed and negative results wereobtained, these results are not conclusive due tothe lack of a positive control. A positive control wasdifficult to obtain as there were no reported casesof APV infection in South Africa at the time of study.This study then does not exclude the possibility ofa concurrent infection of PBFDV and APV in thebirds tested.

The method evaluated in this study for the collec-tion of blood using filter paper was successful anddid not have any negative effects on downstreamprocesses. The results were reproducible and thePCR amplification was comparable to that per-formed using frozen blood samples. This method issimple, easy, does not require large amounts ofblood (thus less stressful to the birds) and the sam-ples do not require the use of anticoagulants, donot have to be frozen and are easy to transport.Most importantly however, it does not affect down-stream processes. With all these advantages it isevident that the implementation of this method ofsample collection is a suitable replacement to themore traditional methods of blood collection.

In conclusion, this study has shown the occurrenceof PBFDV infection in ring-neck parakeets andbudgerigars in South Africa by establishing andoptimising a reliable PCR diagnostic test withreproducible results. The primers that weredesigned during this project were successful inproducing the amplicon of expected size whenused in the PCR test. Genetic variation betweenisolates from ring-neck parakeets and budgerigarsis evident as well as genetic differences betweenisolates within individuals of the same species.However, the significance of these differences toPBFDV antigenicity, pathogenicity and other phys-iochemical characteristics must be investigated ifan effective vaccine is to be produced. Blood sam-ple collection on filter paper is easy to use, is less

stressful to the birds and does not impact negativelyon further processes.

ACKNOWLEDGEMENTS

We appreciate the help of Michel Labuschagne ofthe University of the Free State in the practicalwork. We are grateful to Sam Rosslee, Pierre Swartand G. B. Swart for supplying blood samples and toDr F. Potgieter of the University of the Free Statefor his assistance with the care of the birds.

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SEXTON, N., PENHALE, W.J., PLANT, S.L. & PASS, D.A.1994. Use of goose red blood cells for detection of infectionwith psittacine beak and feather disease virus by haemag-glutination and haemagglutination inhibition. AustralianVeterinary Journal, 71:345–347.

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Psittacine beak and feather disease virus in South Africa